Intestinal stem cells (ISCs) mediate the regenerative response that follows injury to the gut, though the mechanisms that regulate these essential cells remain poorly understood. In the adult, Wnt-responsive Wnt(+) Lgr5+ ISCs are required for intestinal maintenance, but these cells are exquisitely sensitive to injury (e.g., irradiation, inflammation, fasting). In contrast, a reserve population of dormant (d)-ISCs, such as those marked by mTert expression, are highly resistant to injury and become activated to restore Lgr5+ ISCs and tissue homeostasis. Our recent data indicate that d-ISCs (unlike Lgr5+ cells) are maintained in a Wnt-unresponsive Wnt(-) state. Similarly, fetal ISCs are Wnt(-) before birth and mature into Wnt(+) ISCs during the perinatal transition from intra- to extra-uterine life in response to unknown factors. Together, these observations suggest that the transition from a Wnt(-) to a Wnt(+) state is a crucial process for the generation of Lgr5+ ISCs during regeneration and development. Our recent analyses, using RNA-seq to compare Wnt(-) and Wnt(+) ISC populations, revealed multiple differentially expressed genes, underscoring that distinct regulatory pathways control these two ISC populations. Precisely how the Wnt(-) state is maintained in d-ISCs and how it impacts their regenerative response is largely unknown. To address this, we propose the following: Aim 1. Define the role of candidate regulatory factors and the identity of long-lived d-ISCs; Aim 2. Determine how the microbiome and/or its metabolites regulate ISC survival; and Aim 3. Investigate the role of human (h)Tert+ cells in intestinal homeostasis. These studies seek to understand the regulatory mechanisms that govern Wnt(-) ISC maintenance and survival and regulate their transition into Wnt(+) ISCs. The successful completion of these studies may lead to targeted new regenerative therapies for patients suffering from inflammatory bowel disease, short gut syndrome, environmental enteropathy and intestinal toxicity resulting from exposure to radiation and chemotherapy.